Method of microtomy utilizing vitreous carbon blade

ABSTRACT

Methods and apparatus for microtomy utilizing a vitreous carbon knife element having particular properties. The knife element may desirably be rendered hydrophilic along at least one surface to facilitate cleaved sample processing, and provides for economical, high quality cleaving of multiple tissue samples.

The present invention is directed to microtomy methods and apparatus,and, more particularly, is directed to methods and apparatus forsectioning of specimen materials such as tissue samples for microscopicexamination.

The main principles of ultramicrotomy were adapted from lightmicroscopy. However, major modifications had to be made with respect toembedding procedures, the manufacture of knives and the construction ofmicrotomes. Much of this development has been the result of empiricalwork and many details of well known steps in conventional sectioningprocedures are not fully understood in theory.

In conventional sectioning procedures for sample sectioning, frozentissue samples, or tissue samples embedded in either a rigid orsemi-solid organopolymeric matrix are rapidly cleaved by means of amicrotome sectioning knife. In order to produce sections of specimensuitable for ultramicroscopic examination, marks on the specimen due toknife edge defects or deformations, should be minimized, and the knifeedge should function to cleave the specimen cleanly. The quality ofmicrotome sections depends to a large extent on the quality andcharacteristics of the microtome knife, and the cutting process is alsoinfluenced by properties of the trough fluid used in the sectioning andsectioned sample transport operations.

Metallic knives such as steel microtome knives were originally used forcutting sections for ultramicroscopic examination by techniques such aselectron microscopy. However, the use of steel microtome knives incutting sections for electron microscopes has substantial disadvantages,especially in the achievement of satisfactory sample surfaces forexamination and in the provision and maintenance of sufficiently sharpknife edges. Substantial effort has been expended in the art to overcomesuch difficulties and to provide cutting methods utilizing materialshaving suitable homogeneity and hardness without excessive brittleness.One significant result of such development efforts has been theprovision of cleaving methods for providing and using glass "knives" bybreaking glass sheets to produce a cleaved cutting edge [Latta, et al.,Use of a Glass Edge in Tissue Sections for Electron Microscopy, Proc.Bio. Med, Vol. 74, pp. 436-439 (1950)]. By providing a series ofstraight parallel scorings at 90° to the long axis of a glass strip,with one central portion of each 2" distance kept free of score marks,1" glass blocks may be produced that can then be cleaved at a 45° angle(to score-mark free corner) thus producing a knife edge length along thethickness dimension of one surface of one triangle (1"×1"×1/4") and aless perfect edge on one edge of the opposite triangle. However, whilesuch glass knives represent a substantial improvement in the art, suchcleaved edges can be used only for a limited time, and for providing alimited number of sectioned samples.

In view of the tendency of such cleaved edges to lose their propertieswith time and/or use, glass microtome knives are conventionally made onsite as needed under conditions of use. In this latter connection,specific jigs have been developed for producing precision glass knivesfor laboratory applications, specifically for cleaving tissue samplesfor microscopy. Examples of such devices are disclosed in U.S. LettersPat. Nos. 3,207,398, 3,494,521 and 3,908,878.

The use of glass knives in microtomy suffer from a number ofdisadvantages. They may be time consuming to produce and, because glassis physically a super cooled liquid, have a very short life. The cleavededges produced by the intersection of the fracture plane with anotherplane at a score-free junction may be sharp initially, but within amatter of hours and without use, the edge will begin to dull due to flowcharacteristics of the glass, and/or its inability to maintain theprecise molecular arrangement that exists at the cleaving edgeimmediately after breaking. Such knives, as indicated, must therefore beproduced at the point of application since their structural longevity isno more than a day or two in their sharpest state. In addition to suchlimitation, glass knives dull quickly in use and may be utilized onlywith difficulty in providing numerous thick sections of hard specimensincluding routinely embedded materials.

In this regard, not only is it desirable to produce thin sections ofhard samples, it is frequently desirable to prepare samples forultramicroscopic examination by cleaving relatively thick sections ofthe specimen material embedded in an organopolymeric material, such as aspecimen having a thickness in the range of 10 to 50 microns, and toreorient and reembed the thick specimen at a different angle. Thereembedded specimen may then be subsequently sectioned to provide thedesired specimen. Glass knives function best when cleaving sections nomore than 2 microns in thickness, but may be utilized to provide a verylimited number of sections per knife when a thickness of about 2 toabout 10 microns is desired. Thicker sections may not be reliably beprovided through the use of glass knives.

In an attempt to overcome the general thin and thick sectioninglimitations of glass knives, knife edges of harder crystalline materialssuch as diamond have been proposed for microtomy purposes[Fernandez-Moran, H. A., A Diamond Knife for Ultra Thin Sectioning, Exp.Cell Research, 5, pp. 225-256 (1953)], and subsequently have achievedsubstantial commercial application. However diamond knives are veryexpensive and difficult to produce thereby limiting their generalapplicability. Further, diamond knife edges are fragile, and sensitiveto impacts and small blows, so that a knife being used for thicksectioning has a shorter life than one being used for thin sectioning.The economic risk of various sample materials represents a substantiallimitation in the use of diamond knives.

Because of the expense and fragility of diamond knives, various effortshave been made to improve the cutting qualities and longevity ofrelatively inexpensive glass knives. For example, efforts have been madeto coat glass knives with materials such as tungsten in an effort toovercome deficiencies of glass [Roberts, Tungsten Coating-A Method ofImproving Glass Microtome Knives for Cutting Ultrathin Sections, Journalof Microscopy, Vol. 103, Pt. 1, pp. 113-119 (1974)]; but such tecnniqueshave not achieved wide acceptance. Still others [Ward, Some Observationson Glass Knife Making, Stain Technology, Vol. 52, pp. 305-309 (1977)],have tried varying the bevel angle, up to 55°, of the glass knife edgeto enhance cleaving capabilities, but succeed only to a limited degree.

However, despite significant need for improved microtomy methods andapparatus, there have been few significant developments in respect ofglass microtomy knives since their introduction in 1950, and glass anddiamond microtomy systems remain as the two principal alternativesavailable for ultramicroscopic sample sectioning.

Accordingly, there is a need for improved methods and apparatus forspecimen sectioning, and it is an object of the present invention toprovide such methods and apparatus. It is further object to providemethods and apparatus for microtomy which permit the cleavage of arelatively large number of samples from a single, relatively inexpensiveknife edge without the need for the frequent manufacture of new knifeedges common to methods utilizing cleaved glass knives. It is anotherobject of the present invention to provide such methods and apparatusfor providing relatively thick specimen sections as well as thinspecimen sections.

These and other objects of the invention will become apparent from thedetailed description and accompanying drawings of which:

FIG. 1 is a perspective view of microtome apparatus utilizing a knifeelement in accordance with the present invention;

FIG. 2 is a perspective view of the knife element of the microtome ofFIG. 1 and another similar element following a controlled fracture stepin the manufacture of such elements;

FIG. 3 is a schematic illustration of the knife element of the microtomeof FIG. 1, in a manufacturing step subsequent to the fracture stepillustrated in FIG. 2; and

FIG. 4 is a photograph by scanning electron microscope of a portion ofthe cutting edge of the vitreous carbon knife element of FIG. 1 at amagnification of 540;

FIG. 5 is a photograph by scanning electron microscope of the cuttingedge of a vitreous carbon microtome knife element produced by machining,at a magnification of 27;

FIG. 6 is a photograph by scanning electron microscope of a portion ofthe cutting edge of the knife element of FIG. 5, at a magnification of2700, 100 times the magnification of FIG. 5; and

FIG. 7 is a diagram for describing radius of curvature determination.

Generally in accordance with the present invention, methods andapparatus for cleaving specimen materials for microscopic examinationare provided utilizing microtome knife elements of particular design andcomposition, as well as methods of manufacture of such vitreous knifeelements.

In this connection, microtome knife elements are provided in accordancewith the present invention which comprise a vitreous carbon body ofparticular physical property parameters, and having two intersectingsubstantially plane surfaces which intersect along a substantiallylinear intersection edge. The plane surfaces should best intersect at anangle in the range of from about 35° to about 60°, and preferably in therange of from about 40° to about 50°, to form a high performancemicrotome knife edge. At least one of the intersecting plane surfacesadjacent the microtome knife edge may be provided with a hydrophylicsurface, as will be described in more detail hereinafter. As indicated,the microtome knife element utilized in accordance with the presentinvention comprises a vitreous carbon body. Though polymeric carbon is abetter term for this material, due to some of this carbon's propertiesit has been designated vitreous carbon. The vitreous carbon should bestbe an isotropic for certain manufacturing procedures for microtomeknives involving controlled fracture of the material.

Vitreous carbon is a nongraphitic carbon material which may be formed bycontrolled heating of selected polymeric precursors in accordance withknown procedures which generally involve slow carbonization of a formedarticle under conditions which permit diffusion of pyrolysis productswithout disruption of the physical integrity of the artifact, and whichis generally accompanied by a larger, but predictable contraction in thesize of the formed artifact [Jenkens, et al., Polymeric Carbons-CarbonFibre, Glass and Char, Cambridge University Press (1976)]. Vitreouscarbon may be produced in a variety of forms, and is desirably producedin a molded sheet form. The molecular structure of the vitreous carbonis believed to involve carbon atoms joined by strong covalent bonds toform relatively small planar hexagonal arrays plus other carbon arrayswhich are disordered with respect to one another in a turbostraticstructure.

The existence of a cross-linked aromatic structure in the originalpolymer, or during thermal degradation is believed to prevent formationor rearrangement to a full graphite structure on subsequent heating andprovides for the turbostratic structure.

Vitreous carbon knife elements in accordance with the present inventionshould generally be provided from vitreous carbon material having adensity of at least about 1.35 g/cc and typically have a bulk density ofabout 1.45 g/cc. The density will generally be less than about 1.5 g/cc,but it should be noted that the inclusion of carbide forming elementsmay increase the density of a vitreous carbon material.

The physical properties of the vitreous carbon material are important inthe provision of microtome knives. In this connection, the vitreouscarbon should have a compressive strength of at least about 90,000pounds per square inch, and will generally be in the range of from about90,000 to about 140,000 pounds per square inch. The vitreous carbonshould also have a tensile strength of at least about 25,000 pounds persquare inch, and will generally be in the range of from about 25,000 toabout 35,000 pounds per square inch at 20° C. The material shouldfurther have a Young's modulus of at least about 3×10⁶ pounds per squareinch (e.g., in the range of 3-4×10⁶ psi), and a hardness of at least 7on mohs scale. Accordingly, the vitreous carbon material utilized in theknife elements herein is a very hard material which will scratch mostforms of siliceous glass. It is further important that the vitreouscarbon be highly uniform in structure, and in this connection shouldbest be free of crystalline inclusions, porosity or other structuraldefects. In this connection, the vitreous carbon should best have apermeability of less than about 2.5×10⁻¹¹ cm² /sec (helium) and aporosity of less than about 0.05. The vitreous carbon materials shouldbe substantially non-graphitic and homogenous in composition and in thisconnection, the x-ray crystallite size L_(c) of the vitreous carbonshould best be less than about 26 A and more preferably less than about24 A. The thermal conductivity of the vitreous carbon may desirably beat least about 0.01 cal/cm.sec.°C.

As indicated, the vitreous carbon used for knife manufacture by fracturemethods is desirably substantially free of crystalline defects, and inthis connection, it is desirable to use very high purity polymerprecursors which are substantially free of components which induce orprovide carbon (graphite) or carbide crystallization. However, thevitreous carbon may be reacted with various carbide forming elements tomodify the properties of the vitreous carbon. Such reaction may becarried without substantial graphite formation and the materials may becombined within the turbostratic structure of vitreous carbon withoutmerely forming an external deposit. In this regard, a vitreous carbonknife element may be reacted with carbide forming elements such assilicon, boron, tungsten, tantalium, titanium, zirconium, hafnium,vanadium, niobium, chromium, molybdenum, and mixtures thereof withoutsubstantial change in the shape of the knife edge, by selecting avolatile compound (such as hydride) of the carbide forming elements andreacting this compound in the vapor phase with the vitreous carbonmicrotome knife at a suitably elevated temperature.

Turning now to the drawings, various aspects of the present inventionwill be more particularly described with respect to the microtomeapparatus illustrated in FIG. 1. The apparatus 10 is of generallyconventional design comprising an object holder assembly 11 adapted tosecure a specimen 1 for sample preparation. The microtome apparatus 10further comprises a knife holder assembly 12 of conventional design ofthe type utilized for holding glass and diamond knife elements, andwhich is adapted to secure in mounted relationship thereto anunconventional knife element 30 of particular specification inaccordance with the present invention. The illustrated holder assembly12 comprises a steel yoke 13 with a soft plastic blunt end which restsagainst the knife, and provides a slot 14 at its midpoint which issufficiently wide to accomodate the thickness of the vitreous carbonknife element 30. The vitreous carbon knife element is mounted in theholder assembly 12 between the yoke 13 and the knife slot 14 and held inalignment with the yoke 13 when tightened. The knife holder 12, with thevitreous carbon knife in place, is then placed in final knife angleadjustment by being secured to 15 of the ultra microtome. Theillustrated vitreous carbon knife element has a substantially linearknife edge 31 of extreme sharpness which has exceptional capacity forsample cleavage. In the illustrated embodiment 10, at least one planesurface 22 of the knife 30 is rendered hydrophilic and a water trough isprovided along the surface 22 to float cleaved sample sections off theedge of the knife in accordance with conventional practice.

In operation, the object holder is moved toward the knife element 30 andthe sample impacts the knife edge 31 to cleave sample tissues from thesample object.

The sample may be of the organopolymeric impregnated type in which atissue specimen has diffused thereinto an organopolymeric precursor suchas an acrylic monomer or epoxy resin precursor, which is subsequentlypolymerized to provide a rigid and relatively hard sample specimen forcleavage. The forcing of the object against the edge of the knifeelement 30 may generate immense pressures and mechanical strains at theknife edge, and the knife 30 must be capable of repeatedly withstandingsuch conditions. While individual knife elements vary, such conditionsnormally would require the changing of a cleaved glass knife elementafter, for example, less than about 10 specimen sample sections ofconventional thickness in a range of less than 2 microns and about 5microns of 2-10 microns thickness. Substantial difficulty may beexperienced with conventional glass knifes in efforts to cleave samplesof greater thicknesses, such as from about 10 to 50 microns inthickness. However, the vitreous carbon knife element 30 readily andrepeatedly cleaves relatively thick organopolymeric impregnated specimensamples in the range of from about 10 to 50 microns of thickness and isutilizable in the cleavage of a relatively large number of specimens,for example, in excess of 100 specimens without a change in quality.

As indicated, the knife elements provided in accordance with the presentinvention are manufactured of vitreous carbon, and may be provided usingslightly modified equipment similar to that used in the manufacture ofglass microtome knife elements. In this connection, the microtome knifeelements may be manufactured by providing a suitable vitreous carbonsheet having substantially flat parallel surfaces, scoring the sheetalong a first line, fracturing the sheet along the first lineorthagonally to the parallel surfaces to form a first substantially flatcleaved surface, scoring the sheet along a second line intersecting thefirst scored line and fracturing the sheet along the second line to forma substantially flat cleaved surface orthagonal to said sheet surface,free of score marks and intersecting the first cleaved surface to form amicrotome knife edge.

FIG. 2 illustrates in perspective view the knife element 30 which hasbeen broken from a scored vitreous carbon plate 31 having flat, smooth,parallel surfaces 32, 33 and which has previously been fractured along aline 34 to form a substantially planar fracture surface 35 perpendicularto the surfaces 32, 33. The plate 31 is provided from a commerciallyavailable VITRECARB vitreous carbon sheet having a thickness of 0.25inch and a length of about 2 cm. which is manufactured by FluorocarbonCompany of Anaheim, Calif. and has a density of 1.47 g/ml, apermeability of less than 2.5×10⁻¹¹ cm² /sec, a porosity of less than0.05 percent, a thermal conductivity in the range of 0.01 to 0.02cal/cm/sec/°C., a compressive strength in the range of 90,000 to 140,000psi, a tensile strength in the range of 25,000 to 35,000 psi at 20° C.,and a Young's modulus of 3-4×6¹⁰ psi. The plate 31 is substantially purecarbon (about 2 ppm impurities) which is substantially free ofcrystalline carbide inclusions.

The plate 31 is fractured along diagonal score line 36 to form afracture plane 37 which provides a microtome knife edge 38 at itsintersection with the fracture plane 35 and leaving a shelf 39 on theopposite triangle. The angle formed by the intersection of the fractureplanes 35, 37 is typically in the range of from about 45° to about 55°,but may be varied within a broad range. The intersection 38 of thefracture planes 35, 37 forms an extremely sharp substantially linearedge, which has a radius of curvature of less than about 5μ. By radiusof curvature is meant, the radius of curature R of a plane curve at anypoint P (FIG. 7) is the distance, measured along the normal, on theconcave side of the curve, to the center of curvature, C, this pointbeing the limiting position of the point of intersection of the normalsat P and a neighboring point Q, as Q is made to approach P along thecurve.

While the illustrated knife edge 38 is manufactured by cleavagetechniques as previously described, vitreous carbon microtome knives maybe provided by grinding and lapping procedures and polished to produce avery sharp and substantially linear knife edge.

In this connection, a vitreous carbon knife is prepared by first cuttinga 1"×1"×1/4" square at 45° angle from corner to corner using a standardmechanical diamond saw to form triangles of equal dimensions. Next roughgrinding is performed on a standard low speed lapping machine to formthe cutting edge. This is a step wise procedure starting with 250 microndiamonds embedded in a metal disk, going down to 15 microns diamonds toachieve a straight, linear edge. Further lapping and polishing is nowperformed on a standard low speed lapping machine with a furtherreduction in diamond size, down to 1 micron. The lap used, being made ofsoft metal, is prepared in a typical fashion which achieves a straight,linear edge on the vitreous carbon knife. A combination of polishingmaterials such as silicon oxide and aluminum oxide, together with"carriers" of water, detergents and oils are used to achieve the finaledge. FIGS. 5 and 6 are photographs of a knife element produced by theabove procedures, which were taken at a magnification of 27 times and2700 times respectively to show a substantially linear and defect freeknife edge. Since all three surfaces of the triangle are lapped andpolished, it is possible to produce two cutting edges on one knife,whereas a cleaved vitreous carbon knife only has one cutting edge.

Such procedures may advantageously provide knife edges of substantiallength which could be used on histological microtomes which require 25mm to 38 mm knife lengths or longer; while utilization of fractureprocedures tends to limit the maximum knife edge length to the thicknessof the carbon plate, which in turn is limited by the vitreous carbonmanufacturing process.

Vitreous carbon is a hydrophobic material, and in order to provide foruse of the knife edge 38 with water for cleaved sample handling inaccordance with conventional sample handling techniques, at least onesurface adjacent to the edge may be rendered hydrophilic, although itwill be appreciated that such treatment should not substantially degradethe knife edge sharpness.

Hydrophilic properties may be provided by acceptance of theelectrostatic charge on the vitreous carbon surface, and in this regard,FIG. 3 illustrates the changing of the surface properties of vitreouscarbon knife element 30 by ionization treatment. In this connection, itis important to note that the cutting surface of a vitreous carbon knifemust be rendered hydrophilic or it becomes almost impossible to use as asectioning tool. Due to the physical chemistry of the surface, thevitreous carbon attracts and acquires an electronic charge which rendersit hydrophobic and unuseable. Therefore one must deionize the surface,as described below, or use other suitable procedures, which may beutilized to treat the vitreous carbon microtome surface, if desired. Asshown in FIG. 3, the preformed knife element 30 may be placed on thestage 42 in an evacuated chamber (e.g., at a vacuum of 150 millitorr) ofa vacuum evaporator using a filament voltage to an ionization probe 44of about 40 volts for 2-5 minutes to render the microtome surfacehydrophilic.

In accordance with the present invention, vitreous carbon microtomeknives of high sectioning capacity and extreme sharpness may beprovided. In this connection, FIG. 4 is a scanning electron microscopephotomicrograph of a portion of the knife edge 28 of the microtome knife20 following cleavage and ionization treatment. The photomicrograph ofFIG. 4 is taken at a magnification of 540 times and illustrates theuniformity of the cutting edge, as well as the sharpness of the edgewhich may be achieved.

In order to demonstrate the performance of the microtome methods andapparatus in accordance with the present invention, a series of sectionsof various tissue samples and of varying thicknesses are taken over aperiod of six weeks using a Sorvall® MT-2B UltraMicrotome in which ismounted a vitreous carbon microtome knife manufactured in accordancewith the previous disclosure.

The following table presents the data in connection with the variousruns:

                  TABLE 1                                                         ______________________________________                                        SECTIONING RUN                                                                WITH HYDROPHOBIC KNIFE ELEMENT                                                (Knife Not Treated By Ionization)                                                           Section Quality                                                       Estimated     Acceptable                                                Sec-  Section       Publisher Part    Unac-                                   tion #                                                                              Thickness (μM)                                                                           Quality   Acceptable                                                                            ceptable                                ______________________________________                                        1     Cannot Determine              X                                         2     "                             X                                         3     "                             X                                         4     "                             X                                         5     "                             X                                         6     "                             X                                         7     "                             X                                         8     "                             X                                         9     "                             X                                         10    "                             X                                         RUN TERMINATED                                                                ______________________________________                                         NOTE:                                                                         Due to hydrophobic properties of the knife, quality sections were             unattainable.                                                                 SPECIMEN TYPE: Anterior angle/animal eye                                      EMBEDDING MEDIA: Epon (Epoxy resin)                                      

As a comparison, a conventional glass microtome knife is freshlyprepared and is used in the microtome. The glass knives are unable tosatisfactorily cleave specimens greater than 10 microns in thickness andmust frequently be replaced with a new glass knife after taking about 5full-thickness sections of dimensions in the range of 2-10 microns.

                  TABLE 2                                                         ______________________________________                                        SECTIONING RUN                                                                WITH HYDROPHILIC KNIFE ELEMENT                                                (Knife Treated By Ionization)                                                               Section Quality                                                         Estimated   Acceptable                                                        Section     Publisher Part    Unac-                                   Section #                                                                             Thickness (μM)                                                                         Quality   Acceptable                                                                            ceptable                                ______________________________________                                        1        4          X                                                         2        2          X                                                         3       25                            X                                       4       10                    X                                               5       10                    X                                               6       10                    X                                               7       10          X                                                         8       10          X                                                         9       10          X                                                         10      10          X                                                         11      15                    X                                               12      15                    X                                               13      15                    X                                               14      15          X                                                         15      15                    X                                               16      25                    X                                               17      25                    X                                               18      25                    X                                               19      15          X                                                         20       5          X                                                         21      10                            X                                       22      10          X                                                         23      30          X                                                         24      30          X                                                         25      15          X                                                         26      10          X                                                         27      10          X                                                         28      10          X                                                         29      10          X                                                         30      25          X                                                         31      25          X                                                         32       5          X                                                         33      15          X                                                         34      20          X                                                         35      30          X                                                         36      30          X                                                         37      30          X                                                         38      40                    X                                               39      40          X                                                         40      40          X                                                         41       40+        X                                                         42       40+        X                                                         43       40+        X                                                         44      40          X                                                         45      40          X                                                         46      40          X                                                         47      40          X                                                         48      40          X                                                         49      40          X                                                         50      30          X                                                         50      30                                                                    51      30                    X                                               52       2          X                                                         53       50+        X                                                         54      1-2         X                                                         55      1-2         X                                                         56      1-2         X                                                         57      1-2                   X                                               58      1-2         X                                                         59      1-2         X                                                         60      1-2         X                                                         61      1-2         X                                                         62      30          X                                                         63      30                            X                                       64      30                            X                                       65      20                    X                                               66      30                    X                                               67      30          X                                                         68      30          X                                                         69      30                    X                                               70      30          X                                                         ______________________________________                                         NOTE:                                                                         This knife sample is approximately 5 months old, and will still cleave        acceptable samples                                                            SPECIMEN TYPE: Same as in Table 1                                             EMBEDDING MEDIA: Same as in Table 1                                      

The deterioration of sectioning capability of the glass knivesapparently may represent no more than subtle or small scale changes inthe knife structure. In this connection, comparison of a scanningelectron microscope micrograph, examined at 540x, of a glass knife afterfailure through use after cleaving 47 specimens of 1000 A-30μ thicknessof which time the knife would no longer thin or thick section.Comparison with a similar micrograph of a glass knife, made immediatelyafter cleaving that knife, does not reveal any substantial differencesin the edge appearance in the scanning electron micrograph. On the otherhand, vitreous carbon knives may have visible edge defects producedthrough use, and still be capable of cleaving specimen samples.

It will be appreciated by those skilled in the microtomy art that thepresent disclosure has provided improved methods and apparatus formicrotomy.

While the present invention has been particularly described with respectto certain specific embodiments, various modifications, adaptations andvariations will be apparent based on the present disclosure, and areintended to be within the spirit and scope of the present invention.

Various of the features of the invention are set forth in the followingclaims.

What is claimed is:
 1. A method for sectioning a sample for microscopicexamination comprising the steps of providing a sample to be sectioned,providing a microtome knife element comprising a vitreous carbon bodyhaving two intersecting substantially plane surfaces which intersectalong a substantially linear intersection edge, and cleaving said sampleagainst said edge to provide a cleaved specimen of said sample.
 2. Amethod in accordance with claim 1 wherein said vitreous carbon body hasa hardness of about 7 mohs.
 3. A method in accordance with claim 1wherein said sample is an organopolymeric impregnated sample and whereinsaid cleaved specimen has a thickness in the range of from about 0.1 toabout 50 microns.
 4. A method in accordance with claim 1 wherein atleast about 100 specimens are cleaved against a specific region of saidedge.
 5. A method in accordance with claim 1 wherein said planesintersect at an angle in the range of from about 40° to about 60°,wherein said edge is at least about 0.25 cm in length, and wherein saidedge has a radius of curvature of less than about 5μ.
 6. A method inaccordance with claim 1 wherein said vitreous carbon has a density of atleast about 1.35 g/cm³, a compressive strength of at least 90,000 psi, ahardness of at least 7 on mohs scale, and a porosity of less than 0.05percent.
 7. A method in accordance with claim 1 wherein at least one ofsaid vitreous carbon body plane surfaces is hydrophilic.
 8. A method inaccordance with claim 1 wherein said vitreous carbon body is reactedwith a carbide forming element to provide a mohs hardness in excess of 7and less than
 10. 9. A method in accordance with claim 1 wherein saidvitreous carbon body has a density of at least about 1.35 grams per cc,a permeability of less than about 2.5×10¹¹, a porosity of less thanabout 0.05%, a compressive strength of at least about 90,000 psi, atensile strength of at least about 25,000 psi and a mohs hardness of atleast about 7.